The present invention relates to a protease inhibitor of the insect Manduca sexta, its purification, cloning of a DNA sequence encoding the inhibitor, modifications to change inhibitor specificity, transgenic plants carrying and expressing said DNA sequence and insect resistance conferred thereby on said transgenic pants.
The serpins are a superfamily of serine proteinase inhibitors. Human plasma contains serpins which are similar in amino acid sequence and mechanism of inhibition, but differ in their specificity toward proteolytic enzymes. The serpin superfamily includes proteins of about M.sub.r =50,000-100,000, which function in regulation of blood clotting (antithrombin-III, heparin cofactor-II, antiplasmin, protein C inhibitor), complement activation (C1 inhibitor), and proteinases released from neutrophils (.alpha..sub.1 -antitrypsin, .alpha..sub.1 -antichymotrypsin). The serpin superfamily also includes endothelial plasminogen activator inhibitor, glia-derived nexin, mouse contrapsin, ovalbumin, angiotensinogen, barley endosperm protein Z, and cowpox virus 38-kDa protein. Comparisons of the amino acid sequences of these individual serpins reveal a sequence identity of about 20-30%, with the greatest sequence conservation appearing at the COOH-terminal half of the proteins.
In contrast, much less is known about proteinase inhibitors from invertebrates. Most of the proteinase inhibitors isolated from invertebrates have been in the low M.sub.r range of about 5,000-15,000. A M.sub.r =155,000 proteinase inhibitor has been isolated from crayfish plasma and an .alpha..sub.2 -macroglobulin-like proteinase inhibitor has been isolated from hemolymph of the American Lobster.
The only proteinase inhibitors isolated from invertebrates which are similar in size and characteristics to the serpins are a trypsin inhibitor (M.sub.r =42,000) and a chymotrypsin inhibitor (M.sub.r =43,000) which have been isolated from the hemolymph of the silkworm Bombyx mori. Sequences of the amino acids of these two silkworm proteinase inhibitors have revealed 56% amino acid identity with Manduca alaserpin (Takagi et al. (1990) J. Biochem. 108:372-378; Sasaki (1991) Eur. J. Biochem. 202:255).
In higher plants the natural repertoire of weapons available to fight insect predation are believed to include protease inhibitors (PIs). Known for some time (Reed and Haas, 1938 Cereal Chem. 15:59-68), diverse types of plant PIs are thought to provide protection to the tissues containing them (Richardson, 1977 Phytochemistry 16:159-169). Under certain conditions, insecticidal effects result from the addition of PIs to the artificial diets of insects (Steffens et al., 1978 J. Agar Food Chem. 26:170-176; Gatehouse et al., 1980 Phytochemistry 19:751-756; Broadway and Duffy, 1986a J. Insect Physiol. 32:827-833; Broadway and Duffy 1986b Entomologia Experimentalis et Applicata 41:33-38). Under field conditions, Vigna unguiculata (cowpea) varieties with high levels of trypsin inhibitor are more resistant to insect damage than varieties with low levels of the trypsin inhibitor (Gatehouse et al., 1980). In response to localized tissue injury, PI induction occurs on the wounded surface and continues through the remainder of the plant (Green and Ryan, 1972 Science 175:776-777).
The mechanism of PI action that accounts for insect toxicity cannot be easily explained. The inhibitor may arrest protein digestion, reducing essential amino acid levels and restricting insect growth and development. An important secondary effect caused by PIs may be the loss of valuable ions of the insect gut, in response to protease overproduction in the presence of PIs (Liener et al., 1980 Toxic Constituents of Plant Foodstuffs 2 edn., Academic Press, New York). Other environmental factors must also play a role in PI toxicity, as the inclusion of 10% cowpea trypsin inhibitor into artificial feeding tests are nontoxic (Pusztai et al., 1992 Brit. J. Nutrition 68:783-791). However, Boulter et al., EPA 0135343, published Mar. 27, 1985, reported that while cowpea PI was claimed to be effective when applied to edible plant parts, other trypsin inhibitors, e.g., those from soybean and lima bean, had no corresponding effect. The cDNA of this cowpea PI, a Bowman-Birk type inhibitor, was placed under the control of the 35S promoter of Cauliflower Mosaic virus (CaMV), and the 3' terminator of the nopaline synthase (Nos) gene. Cowpea PI was selected because it was previously known to have insecticidal activity. In numerous transgenic plants, the cowpea PI was expressed and accumulated anywhere from 0-1% of the total protein. Confirmation of PI production was via Western and enzyme activity assays. Bioassay of clones with first instar larvae of H. virescens were also conducted. In this case this trypsin inhibitor was effective in protecting against damaging insects. Insect survival was nearly 50% of the control (nontransformed) plant. (See also Hilder, V. A. et al. (1987) Nature 330:160-163.) Tomato inhibitor II (anti-trypsin) also lowered the impact of predators on crop yield by decreasing the larval weight by as much as 15% (Johnson et al., 1989 Proc. Natl. Acad. Sci. USA 86:9871-9875). In the same study, however, tomato inhibitor I (a strong anti-chymotrypsin and weak anti-trypsin inhibitor) was ineffective in protecting against insect predation, and instead supported larval growth as well as did the control (nontransformed) plants.
Inhibitors of the Bowman-Birk type are relatively small (about 70 amino acids length) and multiply cross-linked with disulfite bridges. The Bowman-Birk inhibitors often display dual specificity, inhibiting, e.g., both trypsin and chymotrypsin. The inhibitors are found in many legume varieties. (See Ikenaka, T. and Norioka, S. (1986), in Proteinase Inhibitors, Barrett, A. J. and Salvesen, G. (eds.), Elsevier, Amsterdam, pp. 361-374).
No pattern has emerged to establish which inhibitors have a protective effect and which do not. Inhibitor specificity does not appear to be the only factor, since some trypsin inhibitor are effective while others are not. Little is known regarding the protective effects of chymotrypsin inhibitors or of elastase inhibitors. Inhibitors of the serpin superfamily have not previously been reported to have any protective effect in plants. Other factors affecting protection, such as pH optima and specific salt requirements, have not been systematically studied. Furthermore, experimental data over a range of test species is lacking. Protective effects have been demonstrated for only a few insect species.
Insect pests continue to cause significant crop losses each growing season. Although chemical insecticides are important for crop protection, concerns about toxicity and development of resistance among target populations have prompted a demand for alternative pest-control strategies. One such strategy is the use of insecticidal proteins, either applied on the plant surface or synthesized internally by plants genetically modified to express the protein. The toxins of various strains of Bacillus thuringiensis have been successfully employed, both topically and transgenically. Although protease PIs have been reported to protect plants against insect damage, both applied topically and expressed transgenically, the results have not been uniform, depending both on the target insect and on the specific PI employed. Nevertheless, certain PIs, such as the M. sexta serpin described herein, provide protection for certain crops and pests not otherwise controlled by prior art means.
The whitefly, Bemisia tabaci, has recently emerged as a major pest of vegetable crops and cotton in the far West and Southwest (see Henneberry, T. J. et al. (1992) "First Annual Review of the 5-year National Research and Action Plant for Development of Management and Control of the Sweet Potato Whitefly" USDA-ARS Bulletin. The insect is largely resistent to currently licensed insecticides. Various B. thuringiensis toxins are similarly ineffective, either as applied to plant surfaces or as transgenes. The whitefly feeds on phloem sap, whereas the B. thuringiensis toxin of transgenic plants is primarily intracellular.